Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

Abstract

Background and purpose: Fenamates are N-phenyl-substituted anthranilic acid derivatives clinically used as non-steroid anti-inflammatory drugs in pain treatment. Reports describing fenamates as tools to interfere with cellular volume regulation attracted our attention based on our interest in the role of the volume-modulated transient receptor potential (TRP) channels TRPM3 and TRPV4.

Experimental approach: Firstly, we measured the blocking potencies and selectivities of fenamates on TRPM3 and TRPV4 as well as TRPC6 and TRPM2 by Ca(2+) imaging in the heterologous HEK293 cell system. Secondly, we further investigated the effects of mefenamic acid on cytosolic Ca(2+) and on the membrane voltage in single HEK293 cells that exogenously express TRPM3. Thirdly, in insulin-secreting INS-1E cells, which endogenously express TRPM3, we validated the effect of mefenamic acid on cytosolic Ca(2+) and insulin secretion.

Key results: We identified and characterized mefenamic acid as a selective and potent TRPM3 blocker, whereas other fenamate structures non-selectively blocked TRPM3, TRPV4, TRPC6 and TRPM2.

Conclusions and implications: This study reveals that mefenamic acid selectively inhibits TRPM3-mediated calcium entry. This selectivity was further confirmed using insulin-secreting cells. K(ATP) channel-dependent increases in cytosolic Ca(2+) and insulin secretion were not blocked by mefenamic acid, but the selective stimulation of TRPM3-dependent Ca(2+) entry and insulin secretion induced by pregnenolone sulphate were inhibited. However, the physiological regulator of TRPM3 in insulin-secreting cells remains to be elucidated, as well as the conditions under which the inhibition of TRPM3 can impair pancreatic β-cell function. Our results strongly suggest mefenamic acid is the most selective fenamate to interfere with TRPM3 function.

Figure 1

Structures of the fenamates tested. Flufenamic acid (N-[3-(trifluoromethyl)-phenyl]anthranilic acid), niflumic acid (2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid), S645648 (N-(1,3-dimethyl-5-pyrazolyl) anthranilic acid), DCDPC (3′-5-dichlorodiphenylamine-2-carboxylic acid), meclofenamic acid (N-(2,6-dichloro-3-methylphenyl)anthranilic acid), tolfenamic acid (N-(2-methyl-3-chlorophenyl)anthranilic acid) and mefenamic acid (2-(2,3-dimethylphenyl)aminobenzoic acid).

Figure 2

Concentration–response relationship of transient receptor potential (TRP) channel-blocking fenamates. (A) Calcium entry in TRPM3-expressing cells was measured using FLIPR^Tetra and data analysed as described in Methods. Data from a representative experiment show the effect of mefenamic acid on calcium entry in TRPC6-, TRPM2-, TRPM3- and TRPV4-expressing cells upon hyperforin (10 µM), hydrogen peroxide (5 mM), pregnenolone sulphate (35 µM) and 4α-phorbol-didecanoate (5 µM) stimulation respectively. Mefenamic acid was ineffective at suppressing TRPC6-, TRPM2- and TRPV4-mediated calcium entry, whereas the intracellular calcium concentration ([Ca²⁺]_i) remained at the initial basal level in TRPM3-expressing cells upon stimulation with pregnenolone sulphate. The data were calculated from one experiment of at least three experiments performed in quadruplicate per concentration and TRP channel. (B) In transiently transfected HEK cells expressing TRPM3, the application of pregnenolone sulphate (35 µM) results in an instantaneous increase in intracellular calcium. The application of mefenamic acid (30 µM) during the plateau phase results in a rapid decrease in [Ca²⁺]_i to basal levels. Application of vehicle had no effect. Mean value obtained from at least 20 cells of one experiment (out of five independent experiments) is shown. The grey shading represents the SEM for each data point.

Figure 3

Extracellular application of mefenamic acid inhibits TRPM3 currents in transfected HEK293 cells. (A) Current of TRPM3 at membrane potentials of −80 (lower trace) and +80 mV (upper trace) during the application of the TRPM3 activator pregnenolone sulphate (35 µM) and the inhibition of the current after application of mefenamic acid (25 µM). (B) Current–voltage relationship from experiments shown in (A). Mefenamic acid inhibited the inward current almost completely, although a small outward current is still visible. (C) Current recorded from a TRPM3-expressing cell, experiments performed as in (A) with the modification that mefenamic acid (35 µM) was added into the pipette solution. (D) Current–voltage relationship from the experiment shown in (C). Intracellularly applied mefenamic acid had no impact on the pregnenolone sulphate-induced current.

Figure 4

pH-dependence of mefenamic acid on the inhibition of TRPM3-mediated currents. (A) Time course of TRPM3 current at membrane potentials of −80 (lower trace) and +80 mV (upper trace); the pH value of the extracellular solution was 8 before the mefenamic acid (25 µM) was added. (B) Time course of TRPM3 current at membrane potentials of −80 and +80 mV; the pH value of the extracellular solution was 7.4 before the mefenamic acid (25 µM) was added. (C) Time course of TRPM3 current at membrane potentials of −80 and +80 mV; the pH values of the extracellular solution were 6.6 and 6.0 before the mefenamic acid (25 µM) was added. (D) Statistical analysis of experiments performed at pH 8 (n = 9), pH 7.4 (n = 19), pH 6.6 (n = 10) and pH 6 (n = 9). (E) Current–voltage relationship from experiments shown in (A–C). Although the inward currents were almost completely inhibited, the block of the outward current was dependent on the pH values.

Figure 5

Effects of mefenamic acid on pancreatic β-cells. A. Western blot analysis of membranes extracted from INS-1E cells, HEK293 control cells (con) and TRPM3-expressing HEK293 cells (TRPM3). The anti-TRPM3 antibody detected two bands of approx. 150 and 220 kDa (left panel, – peptide). In the presence of the immunogenic peptide the TRPM3-specific signals were absent (right panel, +peptide). B–F. Changes in [Ca²⁺]_i are depicted by the fluorescence ratios F₃₄₀/F₃₈₀ of Fura-2-loaded INS-1E cells. B. Application of mefenamic acid (Mef) did not alter intracellular calcium concentrations. C. Application of capsaicin (100 nM) resulted in a pronounced increase in calcium which was unaffected by mefenamic acid. D. Measurement of [Ca²⁺]_i of INS-1E cells after stimulation with 35 µM pregnenolone sulphate and block with 30 µM mefenamic acid. An additional stimulus with 20 mM glucose (Gluc) in the presence of 35 µM pregnenolone sulphate and 30 µM mefenamic acid triggered a second increase in [Ca²⁺]_i. E. Mefenamic acid had no effect on voltage-gated calcium channels activated by 100 µM tolbutamide (Tolbut) after blocking pregnenolone sulphate (35 µM)-stimulated INS-1E cells. F. Tolbutamide (100 µM) increased the [Ca²⁺]_i of INS-1E cells. The application of 35 µM pregnenolone sulphate further enhanced [Ca²⁺]_i, an effect reversed by mefenamic acid. The black lines indicate mean values from at least four independent experiments with at least 20 cells each. The shaded areas depict the SEM for each data point.

Figure 6

Effects of mefenamic acid on primary mouse pancreatic β-cells. Changes in [Ca²⁺]_i are depicted by the fluorescence ratios F₃₄₀/F₃₈₀ of Fura-2-loaded primary mouse pancreatic islet cells. (A) Measurement of [Ca²⁺]_i after stimulation with 35 µM pregnenolone sulphate and block with 30 µM mefenamic acid. (B) Mefenamic acid (30 µM) had no effect on [Ca²⁺]_i increased by 100 µM tolbutamide (Tolbut). (C) Tolbutamide (100 µM) increased the [Ca²⁺]_i in mouse β-cells. The addition of 35 µM pregnenolone sulphate further enhanced [Ca²⁺]_i, an effect reversed by the application of mefenamic acid. The black lines indicate mean values from at least four independent experiments with at least 20 cells each. The shaded areas depict the SEM for each data point.

Figure 7

Pregnenolone sulphate-mediated insulin secretion is inhibited by mefenamic acid. INS-1E cells were cultured and insulin secretion measured as described in Methods. Test substances were added as indicated. (A) The effect of pregnenolone sulphate and mefenamic acid in the presence of 100 µM tolbutamide. (B) The effect of pregnenolone sulphate and mefenamic acid on insulin secretion induced by 12 mM glucose. Data are means ± SEM of three independent experiments. ***P < 0.001 to 0.5 mM glucose; +++P < 0.001 to 5 mM glucose; ##P < 0.005 and ###P < 0.001 to the respective condition without mefenamic acid; §P < 0.05 and §§P < 0.005 to the respective condition without pregnenolone sulphate; ns, not significant.